Adeno-associated virus materials and methods

ABSTRACT

The present invention provides adeno-associated virus (AAV) materials and methods which are useful for DNA delivery to cells. More particularly, the invention provides recombinant AAV (rAAV) genomes, methods for packaging rAAV genomes, stable host cell lines producing rAAV and methods for delivering genes of interest to cells utilizing the rAAV. Particularly disclosed are rAAV useful in generating immunity to human immunodeficiency virus-1 and in therapeutic gene delivery for treatment of neurological disorders.

The present application is a continuation of U.S. application Ser. No.10/263,127, filed Oct. 2, 2002, now abandoned which in turn is acontinuation of U.S. application Ser. No. 10/077,294, filed Feb. 15,2002, now abandoned which in turn is a continuation of U.S. applicationSer. No. 09/691,604, filed Oct. 18, 2000, which in turn is acontinuation of U.S. application Ser. No. 09/292,703, filed Apr. 15,1999, now abandoned which in turn is a continuation of U.S. applicationSer. No. 09/012,132 filed Jan. 22, 1998, now abandoned which in turn isa continuation of U.S. application Ser. No. 08/466,606 filed Jun. 6,1995, now abandoned which in turn is a continuation-in-part of U.S.application Ser. No. 08/254,358 filed Jun. 6, 1994 now U.S. Pat. No.5,658,785.

FIELD OF THE INVENTION

The present invention generally relates to adeno-associated virus (AAV)materials and methods which are useful for delivering DNA to cells. Moreparticularly, the invention relates to recombinant AAV (rAAV) genomes,to methods for packaging rAAV genomes, to stable cell lines producingrAAV and to methods for delivering genes of interest to cells utilizingthe rAAV.

BACKGROUND

Adeno-associated virus (AAV) is a replication-deficient parvovirus, thesingle-stranded DNA genome of which is about 4.7 kb in length including145 nucleotide inverted terminal repeat (ITRs). See FIG. 1. Thenucleotide sequence of the AAV2 genome is presented in Srivastava etal., J. Virol., 45: 555–564 (1983). Cis-acting sequences directing viralDNA replication (ori), encapsidation/packaging (pkg) and host cellchromosome integration (int) are contained within the ITRs. Three AAVpromoters, p5, p19, and p40 (named for their relative map locations),drive the expression of the two AAV internal open reading framesencoding rep and cap genes. The two rep promoters (p5 and p19), coupledwith the differential splicing of the single AAV intron (at nucleotides2107 and 2227), result in the production of four rep proteins (rep 78,rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possessmultiple enzymatic properties which are ultimately responsible forreplicating the viral genome. The cap gene is expressed from the p40promoter and it encodes the three capsid proteins VP1, VP2, and VP3.Alternative and non-consensus translational start sites are responsiblefor the production of the three related capsid proteins. A singleconsensus polyadenylation site is located at map position 95 of the AAVgenome. The life cycle and genetics of AAV are reviewed in Muzyczka,Current Topics in Microbiology and Immunology, 158: 97–129 (1992).

When AAV infects a human cell, the viral genome integrates intochromosome 19 resulting in latent infection of the cell. Production ofinfectious virus does not occur unless the cell is infected with ahelper virus (for example, adenovirus or herpesvirus). In the case ofadenovirus, genes E1A, E1B, E2A, E4 and VA provide helper functions.Upon infection with a helper virus, the AAV provirus is rescued andamplified, and both AAV and adenovirus are produced.

AAV possesses unique features that make it attractive as a vector fordelivering foreign DNA to cells. AAV infection of cells in culture isnoncytopathic, and natural infection of humans and other animals issilent and asymptomatic. Moreover, AAV infects most (if not all)mammalian cells allowing the possibility of targeting many differenttissues in vivo. Kotin et al., EMBO J., 11(13): 5071–5078 (1992) reportsthat the DNA genome of AAV undergoes targeted integration on chromosome19 upon infection. Replication of the viral DNA is not required forintegration, and thus helper virus is not required for this process. TheAAV proviral genome is infectious as cloned DNA in plasmids which makesconstruction of recombinant genomes feasible. Furthermore, because thesignals directing AAV replication, genome encapsidation and integrationare contained within the ITRs of the AAV genome, the internalapproximately 4.3 kb of the genome (encoding replication and structuralcapsid proteins, rep-cap) may thus be replaced with foreign DNA such asa gene cassette containing a promoter, a DNA of interest and apolyadenylation signal. Another significant feature of AAV is that it isan extremely stable and hearty virus. It easily withstands theconditions used to inactivate adenovirus (56° to 65° C. for severalhours), making cold preservation of rAAV-based vaccines less critical.Finally, AAV-infected cells are not resistant to superinfection.

Various groups have studied the potential use of AAV in treatment ofdisease states. Patent Cooperation Treaty (PCT) InternationalPublication No. WO 91/18088 published. Nov. 28, 1991 and thecorresponding journal article by Chatterjee et al., Science, 258:1485–1488 (1992) describe the transduction of intracellular resistanceto human immunodeficiency virus-1 (HIV-1) in human hematopoietic andnon-hematopoietic cell lines using an rAAV encoding an antisense RNAspecific for the HIV-1 TAR sequence and polyadenylation signal. Thereview article Yu et al., Gene Therapy, 1: 13–26 (1994) concerning genetherapy for HIV-1 infection lists AAV as a possible gene therapy vectorfor hematopoietic stem cells. The use of rAAV vectors as a deliverysystem for stable integration and expression of genes (in particular thecystic fibrosis transmembrane regulator gene) in cultured airwayepithelial cells is described in PCT International Publication No. WO93/24641 published Dec. 9, 1993 and in the corresponding journal articleby Flotte et al., Am. J. Respir. Cell Mol. Biol., 7: 349–356 (1992).Gene therapy involving rAAV in the treatment of hemoglobinopathies andother hematopoietic diseases and in conferring cell-specific multidrugresistance is proposed in PCT International Publication No. WO 93/09239published May 13, 1993; Muro-Cacho et al., J. Immunol., 11: 231–237(1992); LaFace et al., Virol., 162: 483–486 (1988); and Dixit et al.,Gene, 104: 253–257 (1991). Therapeutic gene delivery into glioma cellsis proposed in Tenenbaum et al., Gene Therapy, 1(Supplement 1): S80(1994).

A relatively new concept in the field of gene transfer is thatimmunization may be effected by the product of a transferee gene.Several attempts at “genetic immunization” have been reported includingdirect DNA injection of influenza A nucleoprotein sequences [Ulmer etal., Science, 259: 1475–1749 (1993)], biolistic gun immunization withhuman growth hormone sequences [Tang et al., Nature, 356: 152–154 (1992)and infection with retroviral vectors containing HIV-1 gp160 envelopeprotein sequences [Warner et al., AIDS RESEARCH AND HUMAN RETROVIRUSES,7(8): 645–655 (1991)]. While these approaches appear to be feasible,direct DNA inoculation may not provide long-lasting immune responses andserious questions of safety surround the use of retroviral vectors. Theuse of AAV for genetic immunization is a novel approach that is notsubject to these problems.

An obstacle to the use of AAV for delivery of DNA is the lack of highlyefficient schemes for encapsidation of recombinant genomes. Severalmethods have been described for encapsidating rAAV genomes to generaterecombinant viral particles. These methods all require in trans AAVrep-cap and adenovirus helper functions. The simplest involvestransfecting the rAAV genome into host cells followed by co-infectionwith wild-type AAV and adenovirus. See, for example, U.S. Pat. No.4,797,368 issued Jan. 10, 1989 to Carter and Tratschin, and thecorresponding journal article by Tratschin et al., Mol. Cell. Biol.,5(11): 3251–3260 (1985). This method, however, leads to unacceptablyhigh levels of wild-type AAV. Another general strategy involvessupplying the AAV functions on a second plasmid (separate from the rAAVgenome) that is co-transfected with the rAAV plasmid. See, for example,Hermonat et al., Proc. Natl. Acad. Sci. USA, 81: 6466–6470 (1984) andLebkowski et al., Mol. Cell. Biol., 8(10): 3988–3996 (1988). If nosequence overlap exists between the two plasmids, then wild-type AAVproduction is avoided as is described in Samulski et al., J. Virol,63(9): 3822–3828 (1989). This strategy is inherently inefficient,however, due to the requirement for three separate DNA transfer events(co-transfection of two plasmids as well as infection with adenovirus)to generate rAAV particles. Large scale production of rAAV by thismethod is costly and is subject to variations in transfectionefficiency.

Vincent et al., Vaccines, 90: 353–359 (1990) reports that a cell lineexpressing rep-cap functions could be used to package rAAV. Such methodsstill requires transfection of the rAAV genome into the cell line andthe resulting titer of rAAV reported was very low (only about 10³infectious units/ml). Dutton, Genetic Engineering News, 14(1): 1 and14–15 (Jan. 15, 1994) reports that Dr. Jane Lebkowski of Applied ImmuneSciences manufactures rAAV using chimeric AAV/Epstein-Barr virusplasmids that contain a recombinant AAV genome, the hygromycinresistance gene and the EBV ori P fragment and EBNA gene. The plasmidsare transfected into cells to generate stable cell lines. The stablecell lines are then transfected with wild-type AAV rep-cap functions andinfected with adenovirus to produce rAAV. Like the method of Vincent,the Lebkowski packaging method requires both transfection and infectionevents to generate rAAV particles.

There thus exists a need in the art for efficient methods of packagingrAAV genomes and for specific rAAVs useful as vectors for DNA deliveryto cells.

SUMMARY OF THE INVENTION

The present invention provides recombinant AAV (rAAV) genomes useful fordelivering non-AAV DNA of interest to a cell. The rAAV genomes of theinvention include AAV ITRs flanking non-AAV DNA sequences of interestand lack rep-cap sequences encoding functional rep-cap proteins. If itis desirable to express the DNA of interest as a polypeptide in thecell, the rAAV genome also includes a (constitutive or regulatable)promoter and a polyadenylation signal operably linked to the DNA ofinterest to form a gene cassette. The gene cassette may also includeintron sequences to facilitate processing of the RNA transcript inmammalian host cells. A presently preferred gene cassette includes thefollowing DNA segments: (1) the cytomegalovirus (CMV) immediate earlypromoter, (2) the rabbit β-globin intron, (3) simian immunodeficiencyvirus (SIV) or human immunodeficiency (HIV) rev and envelope (gp160)genes, and (4) the rabbit β-globin polyadenylation signal. The rAAVgenomes of the invention may be assembled in vectors useful fortransfection of cells which are permissible for infection with a helpervirus of AAV (e.g., adenovirus, E1-deleted adenovirus or herpesvirus). Avector of the invention which contains a rAAV genome including theforegoing preferred gene cassette, a neomycin resistance gene, andwild-type AAV rep-cap sequences was deposited in E. coli DH5 cells withthe American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, on Jun. 1, 1994 and was assigned ATCC AccessionNo. 69637.

Presently preferred rAAV genomes include the SIV rev and envelope(gp160) genes, or the HIV rev and envelope genes, as the non-AAV DNA(s)of interest. Also preferred are rAAV genomes which contain sequencesencoding proteins which may ameliorate neurological disorders such as:sequences encoding nerve growth factor (NGF), ciliary neurotrophicfactor (CNTF), brain-derived neurotrophic factor (BDNF), neurotrophins 3and 4/5 (NT-3 and 4/5), glial cell derived neurotrophic factor (GDNF),transforming growth factors (TGF), and acidic and basic fibroblastgrowth factor (a and bFGF); sequences encoding tyrosine hydroxylase (TH)and aromatic amino acid decarboxylase (AADC); sequences encodingsuperoxide dimutase (SOD 1 or 2), catalase and glutathione peroxidase;sequences encoding interferons, lymphokines, cytokines and antagoniststhereof such as tumor necrosis factor (TNF), CD4 specific antibodies,and TNF or CD4 receptors; sequences encoding GABA receptor isoforms, theGABA synthesizing enzyme glutamic-acid decarboxylase (GAD), calciumdependent potassium channels or ATP-sensitive potassium channels; andsequences encoding thymidine kinase. Also contemplated by the inventionare rAAV genomes including globin, oncogene, ras, and p53 sequences.Recombinant AAV genomes including antisense nucleotides that affectexpression of certain genes such as cell death suppressor genes (e.g.,bc1-2) or that affect expression of excitatory amino acid receptors(e.g., glutamate and NMDA receptors) are also contemplated formodulating neurological disorders.

Other DNA sequences of interest contemplated by the invention includesequences from pathogens including: HIV-1 and HIV-2 (sequences otherthan rev and gp160 sequences); human T-lymphotrophic virus types I andII; respiratory syncytial virus; parainfluenza virus types 1–4; measlesvirus; mumps virus; rubella virus; polio viruses types 1–3; influenzavirus types A, B and C; non-human influenza viruses (avian, equine,porcine); hepatitis virus types A, B, C, D and E; rotavirus; norwalkvirus; cytomegaloviruses; Epstein-Barr virus; herpes simplex virus types1 and 2; varicella-zoster virus; human herpes virus type 6; hantavirus;adenoviruses; chlamydia pneumoniae; chlamydia trachomatis; mycoplasmapneumoniae; mycobacterium tuberculosis; atypical mycobacteria; felineleukemia virus; feline immunodeficiency virus; bovine immunodeficiencyvirus; equine infectious anemia virus; caprine arthritis encephalitisvirus; and visna virus.

Cell lines of the invention are stably transfected with both rAAVgenomes of the invention and with copies of the AAV rep and cap genes.Preferred cell lines are mammalian cell lines, for example, HeLa celllines. Infection of the cell lines of the invention with AAV helpervirus results in packaging of the rAAV genomes as infectious rAAVparticles. A presently preferred stable cell line is the A64 HeLa cellline which was deposited with the ATCC on Jun. 1, 1994 and was assignedATCC Accession No. CRL 11639. The present invention also provides stablecell lines containing AAV rep and cap sequences but no rAAV genome.

Recombinant AAV generated by the foregoing packaging process are usefulfor delivering the DNA of interest to cells. In vivo, rAAV may be usedas antisense delivery vectors, gene therapy vectors or vaccine (i.e.,genetic immunization) vectors. Treatment of disease conditionsincluding, for example, AIDS; neurological disorders including cancer,Alzheimer's disease, Parkinson's disease, Huntington's disease, andautoimmune diseases such as multiple sclerosis, trauma, depression,migraine, pain or seizure disorders; adult T-cell leukemia; tropicalspastic paraparesis; upper and lower respiratory tract infections; upperand lower respiratory tract infections; measles; mumps; rubella; polio;influenza; influenza; hepatitis; hepatitis; hepatitis; hepatitis;hepatitis; diarrhea; diarrhea; systemic cytomegalovirus infections;mononucleosis-like illness; systemic Epstein-Barr virus infections;classic infectious mononucleosis; systemic herpes simplex types 1 and 2infections; genital herpes-simplex infections; chickenpox; roseola;febrile illness due to human herpes virus type 6; pneumonia and adultrespiratory distress syndrome; upper and lower respiratory tractinfections; conjunctivitis; upper and lower respiratory tractinfections; upper and lower respiratory tract infections; genital tractinfections; upper and lower respiratory tract infections; pulmonary andextrapulmonary tuberculosis; systemic infections due to atypical,mycobacteria; feline leukemia; feline AIDS; bovine AIDS; equineinfectious anemia; arthritis and encephalitis in goats; and pneumoniaand encephalitis in sheep are contemplated by the invention. As avaccine vector, rAAV delivers a gene of interest to a cell and the geneis expressed in the cell. The vaccine vectors may be used to generateintracellular immunity if the gene product is cytoplasmic (e.g., if thegene product prevents integration or replication of a virus).Alternatively, extracellular/systemic immunity may be generated if thegene product is expressed on the surface of the cell or is secreted.

A host (especially a human host) may be immunized against a polypeptideof a disease-causing organism by administering to the host animmunity-inducing amount of a rAAV of the invention which encodes thepolypeptide. Immunization of a human host with a rAAV of the inventioninvolves administration by inoculation of an immunity-inducing dose ofthe virus by the parenteral route (e.g., by intravenous, intramuscularor subcutaneous injection), by surface scarification or by inoculationinto a body cavity. Typically, one or several inoculations of betweenabout 1000 and about 10,000,000 infectious units each, as measured insusceptible human or nonhuman primate cell lines; are sufficient toeffect immunization of a human host. Virus to be used as a vaccine maybe utilized in liquid or freeze-dried form (in combination with one ormore suitable preservatives and/or protective agents to protect thevirus during the freeze-drying process). For gene therapy (e.g., ofneurological disorders which may be ameliorated by a specific geneproduct) a therapeutically effective dose of a rAAV of the inventionwhich encodes the polypeptide is administered to a host in need of suchtreatment. The use of rAAV of the invention in the manufacture of amedicament for inducing immunity in, or providing gene therapy to, ahost is contemplated.

BRIEF DESCRIPTION OF THE DRAWING

Numerous other aspects and advantages of the present invention will beapparent upon consideration of the following detailed descriptionthereof, reference being made to the drawing wherein:

FIG. 1 is a schematic representation of the AAV genome;

FIG. 2 is a schematic representation of plasmid psub201 which was thesource of AAV2 sequences utilized in the examples;

FIGS. 3A through 3B is a flow diagram of the construction of a rAAVgenome of the invention in vector pAAV/DMV/SIVrev-gp160;

FIG. 4 is a flow diagram of the construction of the vectorpAAV/CMV/SIVrev-gp160/neo/rep-cap useful to generate a stable cell lineproducing rAAV of the invention; and

FIG. 5 is a schematic representation of a method for packaging rAAVutilizing stable host cell lines of the invention.

DETAILED DESCRIPTION OF THE DRAWING

The present invention is illustrated by the following examples relatingto the production and use of rAAV of the invention. Example 1 describesthe construction of a vector including a rAAV genome containing the SIVrev and envelope (gp160) genes, while Example 2 describes theconstruction of a vector including the AAV rep-cap genes and a neomycinresistance gene. Example 3 sets out the construction of a vector to beused to generate stable cell lines producing rAAV from the vectorsdescribed in Examples 1 and 2. The generation of stable cell linesproducing rAAV encoding the SIV rev and gp160 proteins is detailed inExample 4. Example 5 sets out a preferred procedure for purifying rAAVfrom stable cell lines of the invention. Example 6 describes thegeneration of stable cell lines expressing the AAV rep-cap genes.Example 7 presents results of infection of various mammalian cells andcell lines with the rAAV described in Example 4 which show that gp160protein is expressed in the infected cells. Example 8 describes thegeneration of stable cell lines producing a rAAV that includes theβ-galactosidase gene as a DNA of interest and that is useful as apositive control virus for expression of a DNA of interest in targetcells or tissues. Example 9 presents the results of experiments in whichrAAV of the invention was used to express a DNA of interest in vivo.Example 10 describes methods contemplated by the invention forincreasing the titer of rAAV produced by stable cell lines.

EXAMPLE 1

A vector including a rAAV genome containing a SIV rev and envelope(gp160) gene cassette was constructed from an existing plasmiddesignated psub201 [Samulski et al., supra]. FIG. 2 is a diagram ofplasmid psub201 wherein restriction endonuclease sites are shown andabbreviated as follows: P, PvuII; X, XbaI; B, BamHI; H, HindIII; and N,NaeI. The plasmid contains a modified wild-type AAV2 genome clonedbetween the PvuII restriction sites. The DNA sequence of the wild-typeAAV2 genome is set out in SEQ ID NO: 1. The AAV2 sequence was modifiedto include convenient restriction sites. Specifically, two XbaIrestriction sites were added via linker addition at sequence positions190 and 4484. These sites are internal to 191 bp inverted terminalrepeats (ITRs) which included the 145 bp ITRs of the AAV genome. Theinsertion of these sites allows the complete removal of the internal 4.3kb fragment containing the AAV rep-cap genes upon XbaI digestion of theplasmid. In FIG. 2, the 191 bp ITRs are designated by inverted arrows.

The rAAV genome vector of the invention (pAAV/CMV/SIVrev-gp160) wasgenerated in several steps.

First, plasmid psub201 was digested with XbaI and the approximately 4 kbvector fragment including the AAV ITRs was isolated. A CMV geneexpression cassette was then inserted between the AAV ITRs by blunt endligation. The CMV expression cassette was derived as a 1.8 kbXbaI—AflIII DNA fragment from the vector pCMV-NEO-BAM described inKarasuyama et al., J. Exp. Med., 169: 13–25 (1989). Prior to ligation,the molecular ends were filled in using the Klenow fragment of DNApolymerase I. The CMV expression cassette contained a 750 bp portion ofthe CMV immediate early promoter, followed by a 640 bp intron and a 360bp polyadenylation signal sequence which were derived from the rabbitβ-globin gene. Between the intron and poly A sequences were two cloningsites: a unique BamHI site and two flanking EcoRI restriction sites. Theresulting vector was named pAAV/CMV. See FIG. 3A wherein restrictionendonuclease cleavage sites are shown and abbreviated as follows: B,BamHI; E, EcoRI; N, Nael; and P, PvuII.

Second, the pAAV/CMV expression vector was linerized at the BamHI siteand sticky ends were blunted with Klenow. A PCR-generated, 2.7 kb SIVsubgenomic fragment containing the rev and envelope (gp160) sequences[SEQ ID NO: 2, Hirsch et al., Nature, 339: 389–392 (1989)] was clonedinto the blunt-ended BamHI site. The resulting recombinant AAV genomevector, pAAV/CMV/SIVrev-gp160, is 8.53 kb in length. See FIG. 3B whereinrestriction endonuclease cleavage sites are shown and abbreviated asfollows: N, Nael and P, PvuII. The vector contains the following DNAsegments in sequence: (1) an AAV ITR, (2) the CMV promoter, (3) therabbit β-globin intron, (4) the SIV rev and envelope sequences, (5) therabbit β-globin polyadenylation signal, and (6) an AAV ITR. In transienttransfection assays of human 293 cells, this vector resulted in highlevels of expression of the SIV gp160 protein as determined byradioimmunoprecipitation assays using polyclonal sera from monkeysinfected with SIV.

The invention specifically contemplates substitution by standardrecombinant DNA techniques of the following sequences for the SIVrev/envelope sequences in the foregoing vector: HIV-1 rev/envelopesequences (the HIV-1_(MN) rev/envelope sequence is set out in SEQ ID NO:3); nerve growth factor [Levi-Montalcini, Science, 237: 1154–1162(1987)]; ciliary neurotrophic factor [Manthorpe et al., beginning at p.135 in Nerve Growth Factors, Wiley and Sons (1989)]; glial cell derivedneurotrophic factor [Lin et al., Science, 260: 1130–1132 (1993)];transforming growth factors [Puolakkainen et al., beginning at p. 359 inNeurotrophic Factors, Academic Press (1993)]; acidic and basicfibroblast growth factors [Unsicker et al., beginning at p. 313 inNeurotrophic Factors, Academic Press (1993)]; neurotrophin 3[Maisonpierre et al., Genomics, 10: 558–568 (1991)]; brain-derivedneurotrophic factor [Maisonpierre, supra]; neurotrophin 4/5 [Berkemeieret al., Neuron, 7: 857–866 (1991)]; tyrosine hydroxylase [Grima et al.,Nature, 326: 707–711 (1987)]; and aromatic amino acid decarboxylase[Sumi et al., J. Neurochemistry, 55: 1075–1078 (1990)].

EXAMPLE 2

A plasmid designated pSV40/neo/rep-cap which contains the AAV rep-capgenes and a neomycin resistance gene was constructed to be used inconjunction with the rAAV genome vector described in Example 1 togenerate a stable cell line producing rAAV.

A plasmid designated pAAV/SVneo (Samulski et al., supra) was digestedwith EcoRI and BamHI to release a 2.7 kb insert including a 421 bpportion of the SV40 early promoter, a 1.4 kb neomycin resistance gene,and a 852 bp DNA fragment containing the SV40 small t splice site andSV40 polyadenylation signal. This released insert was cloned into theEcoRI and BamHI sites of pBLUESCRIP KS+ (Stratagene, La Jolla, Calif.)to generate the 5.66 kb plasmid pSV40/neo. Next, the approximately 4.3kb DNA fragment containing the AAV rep-cap genes, derived from thedigestion of psub201 with XbaI as described in Example 1, was ligatedinto the XbaI restriction site of pSV40/neo to create the plasmidpSV40/neo/rep-cap (about 10 kb). The construction of this plasmid isdetailed in first half of FIG. 4 wherein restriction endonuclease sitesare shown and abbreviated as follows: B, BamHI; E, EcoRI; HindIII; P,PvuII; N, NotI; RV, EcoRV; and X, XbaI. This plasmid was functional intransient assays for rep and cap activity and was itself ultimately usedto derive stable cell lines (see Example 5 below).

EXAMPLE 3

A final vector to be used to generate stable cell lines producing rAAVwas generated from vector pAAV/CMV/SIVrev-gp160 (Example 1) and plasmidpSV40/neo/rep-cap (Example 2).

The construction entailed removing the neo-rep-cap gene cassette frompSV40/neo/rep-cap and inserting it into a unique NaeI site inpAAV/CMV/SIVrev-gp160 (see FIG. 3B). Specifically, vectorpAAV/CMV/SIVrev-gp160/neo/rep-cap was made by agarose gel band isolatinga 7.0 kb EcoRV-NotI DNA fragment containing the SV/neo and rep-capexpression domains from pSV40/neo/rep-cap. The sticky ends of thefragment were blunted with Klenow and the fragment was ligated into theblunt-ended NaeI site of pAAV/CMV/SIVrev-gp160. See FIG. 4. VectorpAAV/CMV/SIVrev-gp160/neo/rep-cap (ATCC 69637) contains the followingelements: (1) the rAAV genome; (2) AAV rep-cap genes; and (3) theneomycin resistance gene.

EXAMPLE 4

The vector pAAV/CMV/SIVrev-gp160/neo/rep-cap was used to generate stablecells lines containing both the rAAV genome of the invention and AAVrep-cap genes.

HeLa cells at 70% confluency were transfected with 10 μg ofpAAV/CMV/SIVrev-gp160/neo/rep-cap plasmid DNA in 100 mm dishes. Cellswere transfected for 6 hours after formation of DOTAP/DNA complexes inserum minus media as prescribed by the manufacturer's protocol(Boehringer-Mannheim, Indianapolis, Ind.). Following the removal of thetransfection medium, DMEM media containing 10% fetal bovine serum wasadded to the cells. Three days later, media supplemented with 700 μg/mlGeneticin (Gibco-BRL, Gaithersburg, Md.) was used to select for cellsthat stably expressed the neomycin resistance gene. Fresh Geneticincontaining DMEM media was added every four days. Geneticin resistantclones were selected 10–14 days after selective media was added. A totalof fifty-five colonies were selected and transferred to 24-well platesand expanded for further analysis.

The fifty-five neomycin resistant HeLa cell lines were initiallyscreened for functional rep gene activity; twenty-one scored positive.Rep gene activity was assayed by infecting the cell lines withadenovirus type 5 (Ad5). Infection by adenovirus transactivates the repand cap genes. This results in the replication of the rAAV genome andsubsequent encapsidation of these sequences into infectious rAAVparticles. A schematic representation of rAAV production is shown inFIG. 5. Following maximum Ad5-induced cytopathic effect (CPE; roundingof cells and 90% detachment from the culture flask), cell lysates wereprepared and Hirt DNA (low molecular weight DNA) was isolated [Hirt, J.Mol. Biol., 26: 365–369 (1967)]. Southern blot analysis was used tovisualize the synthesis of recombinant AAV (rAAV) replicative forms(single strand, monomeric, and dimeric forms). Control wells notreceiving Ad5 were always negative. Cell lines with high relative levelsof rep gene activity were selected for further study.

To assay for cap gene functionality, cell lines were infected with Ad5and clarified lysates prepared after the development of maximum CPE. Thecell lysates, Ad5, and wild-type AAV were used to infect HeLa cells.Following the development of Ad5 induced CPE (72 hr), Hirt DNA wasisolated and Southern blot analysis performed. Cell line lysates thatgave rise to gp160 hybridizable rAAV (SIV gp160) replicative sequenceswere scored positive for capsid production.

An infectious unit/ml (IU/ml) titer of rAAV produced by each cell linewas derived by co-infecting C12 cells (exhibiting stable rep and capgene expression) with Ad5 and a serial ten-fold dilution of theclarified cell line lysate to be tested. After maximum Ad5-induced CPE,Hirt DNA was isolated and Southern blot analysis performed to detect thepresence of rAAV replicative forms. The end-point dilution that producedvisible monomeric and dimeric replication intermediates was taken as thetiter. Titer estimation was based on two to four replicate experiments.

Results of characterization of eight of the fifty-five cell lines areshown in Table 1 below wherein “ND” indicates a value was notdetermined.

TABLE 1 Cell Line Rep Function Cap Function Titer (IU/ml) A5 ++ + 10⁴A11 ++++ + 10⁵ A15 ++++ + 10⁵ A37 ++++ + ND A60 +++++ − <10¹   A64+++++ + 10⁶ A69 ++ − ND A80 ++++ + 10⁵

Cell line A64 (ATCC CRL 11639) produced a high titer of rAAV (10⁶ iu/ml)in clarified lysates. This titer is approximately 1000-fold higher thanthe titer of rAAV reported by Vincent et al., supra.

The rAAV produced by the various cell lines was also tested for itsability to express SIV gp160 in HeLa cells infected with the recombinantvirus. Concentrated stocks of rAAV produced by the eight stable celllines listed in Table 1 were generated. Cell lysates containing rAAVparticles were subjected to step density gradient (CsCl) purification.After desalting dialysis and heat-inactivation of Ad5, the rAAVparticles were used to infect (transduce) HeLa cells in culture. Twolines of investigation were pursued. First, the transduced cells weretested for the presence of SIV gp160-specific mRNA by performing RT-PCRon total RNA collected 72 hours after transduction. Primers specific forSIV gp160 amplified a predicted 300 bp fragment only in the presence ofreverse transcriptase and Taq polymerase; samples run without reversetranscriptase were uniformly negative. Second, HeLa cells weretransduced with various dilutions of the same rAAV/SIV stock asdescribed above and, at 72 hours post transduction, indirectimmunofluorescence was performed on the infected cells. At all dilutionstested (out to 1:200), cells positive for the SIV gp160 protein weredetected; lower dilutions clearly had more positive cells.

The A64 cell line was tested for wild-type AAV production by a standardmethod. The cell line was infected with adenovirus to produce rAAV as alysate. The lysate was then used to infect normal HeLa cells either: (i)alone; (ii) with adenovirus; or (iii) with adenovirus and wild-type AAV.As a control, HeLa cells were infected with adenovirus and wild-type AAVwithout rAAV. Hirt DNA was prepared and analyzed by Southern blotting(two different blots) for replicating forms of either rAAV or wild-typeAAV. No wild-type AAV was detected in A64 cells not exposed to wild-typeAAV.

Because the present invention involves the establishment of stable celllines containing not only copies of the AAV rep and cap genes, but alsoof the rAAV genome (with ITRs flanking DNA of interest), rAAV isproduced by merely infecting the cell line with adenovirus. Transfectionof exogenous DNA is not required, thereby increasing the efficiency ofrAAV production compared to previously described methods. Othersignificant features of the invention are that no wild-type AAV isproduced and that scale-up for production of rAAV is easy and is limitedonly by normal constraints of cell growth in culture.

EXAMPLE 5

A method to isolate and purify rAAV from stable (producer) cell lineswas developed.

Producer cells (for example, the A64 cells of Example 4) were seeded ata cell density of 3×10⁶ producer cells per 175 cm² surface area ingrowth medium. Cells reached a density of about 8×10⁶ cells after 16–18hours, and were then infected with adenovirus (Ad5) at a multiplicity ofinfection (moi) of 5 for 1–2 hours in growth medium. A 15 ml infectionvolume was used, and after the 1–2 hour infection, 10 ml of growthmedium was added to each flask to obtain a final volume of 25 ml.[Alternatively, Ad5 may be added directly by: removing all but 15 ml ofgrowth medium and adding Ad5 in a volume of 10 ml (diluted in HBSS) togive a final volume of 25 ml.]

Cells were harvested at about 48–60 hours after infection when mostcells released from the flask after a vigorous shake. The cells werethen stained with trypan blue to determine the percentage of viablecells. It is desirable for greater than 80% to be viable. Cells werethen transferred to 250 ml disposable conical bottles (Corning) andpelleted at 1000×g for 15 minutes at 4° C. The resulting supernatant wasremoved saving an aliquot and the cells were suspended in TM buffer (50mM Tris, pH 8.0, and 1 mM MgCl₂) at a density of 5×10⁶ cells/ml. Thecells were subjected to three cycles of freeze/thaw on dry ice withvortexing 2 minutes between each thaw. The lysed cells were than heatedto 56° C. for 30 minutes to 1 hour with vortexing every 7.5 minutesduring the last thaw. Ten percent deoxycholate was added to the lysateto a final concentration of 1%, and the mixture was incubated at 37° C.for 30 minutes with intermittent vortexing to achieve complete lysis. Ifnecessary to achieve complete lysis, the mixture was sonicated 3 timeson maximum setting for 2 minutes each time. A hemocytometer was used toconfirm complete cell lysis. Cell debris was pelleted at 2000×g for 15minutes at 4° C. The rAAV containing supernatant was saved.

The rAAV was isolated using a 1.31 g/ml CsCl cushion. Twenty-one ml oflysate supernatant was layered on a 14 ml CsCl cushion in a SW-28 tube,and spun 16,000 rmp, 10° C. for 16 hours. The resulting supernatant wasaspirated and the rAAV pellet was washed with HBSS to remove residualCsCl. The pAAV pellet was re-dissolved in 20 mM Tris pH 8, 150 mM NaCl,1 mM MgCl₂ (TMN buffer) in the smallest volume manageable (about 500μl/pellet) and let hydrate overnight. It was then heated to 56° C. for30 minutes with vortexing every 5 minutes. At this endpoint, the viruswas dialyzed against the TMN buffer to remove all traces of cesium ifthe virus was not going to be further purified.

The rAAV may be further purified by isopycnic banding. This isappropriate under conditions in which the virus is to be administered invivo. The hydrated rAAV was brought up to a CsCl density of 1.41 g/ml,and then spun in an SW-41 tube at 30K for 48 hours at 10° C. The topportion of the gradient containing adenovirus (density 1.34–1.36 g/ml)was discarded and the remaining portion of the CsCl gradient was dilutedwith TMN down to a buoyant density of less than 1.1 g/ml. rAAV was thenpelleted by an overnight spin at >60,000×g. The rAAV was resuspended ina minimal amount of TMN buffer supplemented with 1% gelatin. Forefficient hydration, the pellet was allowed to sit overnight at 4°. TherAAV was then aliquoted and stored at 20° C.

EXAMPLE 6

Concurrent with the generation of the stable cells described in Example4, stable HeLa cell lines were established by similar methods whichcontained rep-cap genes but no rAAV genome using plasmidpSV40/neo/rep-cap (Example 2). A total of fifty-two neomycin resistantHeLa cell lines were isolated and characterized.

To test for rep gene function, each cell line was infected with Ad5 andsubsequently transfected with pAAV/CMV/SIVrev-gp160. FollowingAd5-induced CPE (72 hr), Hirt DNA was isolated and Southern blotanalysis performed. Rep gene function was scored positive for cell linesthat produced monomeric and dimeric rAAV gp160 sequences. The intensityof autoradiographic signal was used as a relative measure of rep geneexpression (1–5+). Ad5 minus control samples never produced rAAVreplicative forms. Cap gene proficiency was assayed in a similar manner(Ad5 infection and pAAV/CMV/SIVrev-gp160 transfection), except that aclarified cell lysate was prepared after the development of maximum CPE.HeLa cells were then co-infected with a portion of the clarified celllysate, Ad5, and wild-type AAV. Hirt DNA was isolated 72 hours later,and hybridization analysis was used to visualize the existence ofrAAV/gp160 replicative forms (monomeric and dimeric). In the assaydescribed, the C12 cell line yielded the highest relative proportion ofrAAV/gp160/120 sequences.

Results of the characterization assays are presented for eight celllines are presented in Table 2 wherein the abbreviation “ND” indicatesthat a value was not determined.

TABLE 2 Cell Line Rep Function Cap Function C2 +++++ + C12 ++++ +++ C16− ND C18 +++ ND C23 +++ ND C25 +++ − C27 ++ ND C44 ++++ +

There are two principal uses for the stable cell lines expressingrep-cap sequences: (1) generating rAAV particles if the cell lines aretransfected with a rAAV genome and infected with helper virus; and (2)determining rAAV infectious titers. To estimate rAAV infectious titers,these cell lines are co-infected with adenovirus and serial dilutions ofthe rAAV stock. After maximum CPE, Hirt DNA is isolated and replicativerAAV forms are visualized by Southern blot analysis. End point titration(last rAAV stock dilution to give positive hybridization signal) is thenused to determine the infectious titer.

EXAMPLE 7

The ability of the rAAV produced by HeLa cell line A64 to infect(transduce) and produce SIV gp160 protein in various mammalian celltypes in addition to HeLa cells (see Example 4) was assayed. The rAAV(at a multiplicity of infection of approximately 1) was used to infectcells either in a monolayer or in suspension, depending on the celltype. Three days after rAAV infection, the cells were fixed inacetone/methanol and evaluated for the production of gp160 by indirectimmunofluorescence using polyclonal antisera from an SIV-infectedmonkey. The following cells or cell lines were infected and shown toproduce gp160; fetal rat brain cells (neurons and glial cells), mouse3T3 fibroblasts, mouse vagina, human vagina, human colon, human andmonkey lymphocytes and 293 cells. No non-permissive cell type wasidentified. These results demonstrate that the rAAV produced by the A64cell line infects a wide range of mammalian cell types and leads to cellsurface expression of the SIV envelop gene product, gp160, in thetransduced cells.

EXAMPLE 8

Stable cell lines were generated that produced rAAV carrying theβ-galactosidase gene as a gene of interest. These rAAV are useful aspositive control to test for expression of a DNA of interest in a targetcell or tissue.

A vector like pAAV/CMV/SIVrev-gp160/neo/rep-cap was constructed thatincluded a β-galactosidase gene expression cassette (Clontech, PaloAlto, Calif.) containing the human CMV promoter, the E. coliβ-galactosidase gene, and the SV-40 splice/polyadenylation sequenceinstead of the rabbit β-globin intron, SIV rev and envelope sequences,and rabbit β-globin polyadenylation signal between the AAV ITRs. Thisβ-galactosidase gene expression cassette was cloned in between the AAVITRs by standard recombinant methods.

Stable HeLa cell lines which produced rAAV containing theβ-galactosidase gene (rAAV/β-gal) were generated as described in Example4 using the foregoing vector.

EXAMPLE 9

The rAAV/β-gal of Example 8 were used to demonstrate the use of rAAV ofthe invention for gene transfer into the brains of live mice. rAAV/β-galwas injected directly into the brains of mice and the brains were thenexamined for evidence of β-galactosidase activity.

Balb/c mice (n=3; male; 9 months old) were anesthetized and secured on amurine stereotactic platform. Using sterile technique, rAAV/β-gal (1 μlcontaining 3×10⁶ infectious units) was injected into the righthippocampus. Additional mice (n=3) received an injection of diluent ascontrols. One week after injection, mice were sacrificed by cardiacexsanguination followed by sequential infusion of 50 ml of heparinizedphosphate buffered saline, then 50 ml of a mixture of paraformaldehyde(0.5%) and glutaraldehyde (2.5%) in 0.1M phosphate buffer (pH 7.3).Whole brains were removed. post-fixed in the same fixative mixture (2hours) and frozen in O.C.T. Cryostat sections (10 μm) were placed onpoly-L-lysine coated microscope slides and stored at −20° C. Slides werethawed at room temperature, fixed again (5 minutes at 4° C.), washedtwice in PBS, and transferred to X-gal stain (a substrate for theenzymatic activity of β-galactosidase). After incubation overnight at37°, slides were washed twice in PBS, counterstained with nuclear fastred, and examined microscopically for blue-stained cells (cells whereβ-galactosidase was being expressed).

In the brains of the mice injected with rAAV/β-gal, blue-stained cellsin the hippocampus were easily detected upon microscopic examination. Inthe brains of mice injected with diluent (controls), no blue-stainedcells were found.

EXAMPLE 10

Various methods to increase the titer of rAAV generated from stable celllines which involve providing additional AAV rep and cap genes to thecell lines are contemplated by the invention.

In a first method which demonstrates the usefulness of providingadditional rep and cap genes, a producer cell line is transfected with aplasmid containing a helper plasmid carrying AAV rep and cap genes priorto adenovirus infection. Results from experiments in which a rAAV/β-galproducer cell line (H44) was so transfected are presented in Table 3below.

TABLE 3 Treatment Viral Yield IU/cell Fold increase Mock transfection 7× 10⁷ 7 0  50 μg pBS/rep-cap 1 × 10⁹ 100 14 100 μg pBS/rep-cap 8 × 10⁸80 11 150 μg pBS/rep-cap 1 × 10⁹ 110 16

In a second method, the AAV rep and cap genes are placed on a separateplasmid containing an EBV or BPV origin of DNA replication and a drugresistance marker (hygromycin). The plasmid will be transfected into aproducer cell line and new cell lines are then selected on neomycin andhygromycin. This selection pressure will result in stable cell lineswhich contain both rAAV genomes and multiple copies of the AAV rep andcap genes.

In a third method, the AAV rep and cap genes are cloned into a helpervirus genome, for example, into the adenovirus genome in the E3 locationunder the control of the tetracycline operator.

While the present invention has been described in terms of preferredembodiments, it understood that variations and improvements will occurto those skilled in the art. Therefore, only such limitations as appearin the claims should be placed on the invention.

1. A method for producing infectious recombinant adeno-associated viruscomprising the step of infecting a mammalian host cell with a helpervirus of adeno-associated virus, wherein the mammalian host cell isstably transfected with a recombinant adeno-associated virus genome andwith adeno-associated virus rep-cap genes, and wherein the helper viruscontains adeno-associated virus rep-cap genes inserted in its genome. 2.A method for producing infectious recombinant adeno-associated viruscomprising the step of infecting a mammalian host cell with a helpervirus of adeno-associated virus, wherein the mammalian host cell isstably transfected with a recombinant adeno-associated virus genome andwith adeno-associated virus rep-cap genes, wherein the recombinantadeno-associated virus genome comprises adeno-associated virus invertedterminal repeats flanking DNA sequences encoding an immunodeficiencyvirus protein operably linked to promoter and polyadenylation sequences,and wherein the helper virus contains adeno-associated virus rep-capgenes inserted in its genome.
 3. A method for producing infectiousrecombinant adeno-associated virus comprising the step of infecting amammalian host cell with a helper virus of adeno-associated virus,wherein the mammalian host cell is stably transfected with a recombinantadeno-associated virus genome and with adeno-associated virus rep-capgenes, wherein the recombinant adeno-associated virus genome comprisesadeno-associated virus inverted terminal repeats flanking DNA sequencesencoding a polypeptide selected from the group consisting of tyrosinehydroxylase, aromatic amino acid decarboxylase, nerve growth factor,brain derived neurotrophic factor, NT-3, NT-4/5, glial derivedneurotrophic factor and fibroblast growth factor, the DNA sequencesbeing operably linked to promoter and polyadenylation sequences, andwherein the helper virus contains adeno-associated virus rep-cap genesinserted in its genome.
 4. A method for producing infectious recombinantadeno-associated virus comprising the step of infecting a mammalian hostcell with a helper virus of adeno-associated virus, wherein themammalian host cell is stably transfected with a recombinantadeno-associated virus genome and with adeno-associated virus rep-capgenes, wherein the recombinant adeno-associated virus genome comprisesthe cytomegalovirus (CMV) immediate early promoter, the rabbit β-globinintron, the human immunodeficiency virus rev/envelope sequences and therabbit β-globin polyadenylation signal, and wherein the helper viruscontains adeno-associated virus rep-cap genes inserted in its genome.